Application of cyproheptadine hydrochloride in preparation of medicine for preventing and / or treating canine parvovirus disease
Cyproheptadine hydrochloride prevents the virus from invading host cells by inhibiting the transcription and protein expression of the CPV VP2 gene, thus solving the problem of the lack of effective treatment for canine parvovirus disease in existing technologies and achieving a highly efficient therapeutic effect.
Patent Information
- Application Number
- CN202511300955.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2025-12-05
AI Technical Summary
There is currently no application of cyproheptadine hydrochloride for the prevention and treatment of canine parvovirus disease, and CPV virus is highly infectious to host cells, causing severe gastrointestinal and myocarditis symptoms.
Cyproheptadine hydrochloride prevents the virus from invading host cells by inhibiting the transcription and protein expression of the CPV VP2 gene, and is used to prepare drugs for the prevention and treatment of canine parvovirus disease.
Cyproheptadine hydrochloride significantly inhibits CPV replication, significantly improves body temperature, mental state, appetite and fecal condition in affected dogs, with a cure rate of 70%, a significant efficacy rate of 30%, and a total effective rate of 100%. It can be used alone to effectively treat canine parvovirus disease.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of veterinary medicine, in particular to the use of acepromazine hydrochloride in the preparation of a medicine for preventing and / or treating canine parvovirus disease. BACKGROUND
[0002] Canine parvovirus disease (CPD), also known as canine infectious enteritis or canine viral enteritis, is an infectious disease caused by canine parvovirus (CPV), which is characterized by hemorrhagic enteritis and acute myocarditis, and is one of the main factors causing the death of puppies. CPV mainly infects healthy dogs through the digestive tract, and the virus mainly damages the intestinal epithelial cells and myocardial cells after entering the body, and then the body shows corresponding gastrointestinal symptoms and myocarditis symptoms. Clinically, the initial stage is poor mental state, decreased appetite, and body temperature rising to above 40℃, and vomiting. Diarrhea is the main symptom of canine parvovirus disease, and the feces are mainly chocolate color, and are porridge-like or watery. The late stage of the disease develops into severe vomiting, and the diarrhea is mainly watery, with blood in the feces, and is rotten tomato color, and is often accompanied by the characteristic strong odor of canine parvovirus disease, and severe diarrhea will cause the anus of the sick dog to relax.
[0003] The CPV virus particle is small, with a diameter of about 20-24nm, and is a non-evolutionary virus. The CPV virus particle contains three proteins and is a single-stranded DNA virus, which has a cubic symmetry structure and is an isometric icosahedron, composed of a circular-appearing capsid without a membrane, and the capsid is composed of 32 shell particles.
[0004] The CPV genome is composed of a single-stranded negative linear DNA, about 5233 nucleotides. CPV virus mainly contains two open reading frames, one of which encodes two non-structural proteins (NS1, NS2), the NS1 protein can affect the assembly and release of the virus, and the NS2 protein can promote the release of the virus particle from the cell; the other reading frame encodes two capsid proteins (VP1, VP2), of which the former accounts for about 10% of the total, with a molecular weight of about 83KDa, and the latter accounts for more than 90% of the capsid protein, with a molecular weight of about 67KDa. The VP2 protein affects the antigenic properties of CPV, determines the virus coding and recognizes the host cell receptor, and plays an important role in controlling the virus host range and tissue tropism, and each amino acid site mutation may be the cause of affecting the host range of parvovirus; VP3 protein generated by VP2 protein hydrolysis can also be seen in mature virus particles, and the molecular weight of VP3 protein is about 65KDa. The CPV genome includes NS1, NS2, VP1, VP2, and the VP2 gene is most susceptible to mutation, with a mutation rate similar to that of RNA viruses, reaching 2x10 -4 substitutions / sites / year.
[0005] Cyproheptadine Hydrochloride, the molecular formula is C 21 H 26 ClN2·HCl. CAS No. 41354-29-4. Molecular weight is about 381.36 g / mol, which is an antihistamine, an antagonist of 5-hydroxytryptamine (5-HT) and histamine 2, and has obvious anticholinergic and central inhibition effect. It is mainly used for the treatment of various allergic diseases in clinic. But there is no research shows that cyproheptadine hydrochloride has the effect of resisting canine parvovirus. SUMMARY
[0006] The purpose of the present application is to provide the use of cyproheptadine hydrochloride in the preparation of a medicine for preventing and / or treating canine parvovirus disease, in order to solve the problems existing in the prior art. The present application found that the compound cyproheptadine hydrochloride can significantly inhibit the replication of CPV when the cell-based antiviral activity of the virtual screening of the inhibitor targeting CPV VP2 protein was verified, and further found that it is through inhibiting the transcription and protein expression level of CPV VP2 gene. Thus, it can prevent CPV virus from invading host cells, and can be used for inhibiting the infection of CPD. Therefore, cyproheptadine hydrochloride can be used for treating canine parvovirus disease.
[0007] In order to achieve the above-mentioned purpose, the present application provides the following scheme:
[0008] One of the technical schemes of the present application is the use of cyproheptadine hydrochloride in the preparation of a medicine for preventing and / or treating canine parvovirus disease.
[0009] The second technical scheme of the present application is a medicine for preventing and / or treating canine parvovirus disease, which comprises cyproheptadine hydrochloride.
[0010] Based on the above technical scheme, the present application has the following technical effects:
[0011] The present application found that the compound cyproheptadine hydrochloride can significantly inhibit the assembly and release of CPV when the cell-based antiviral activity of the virtual screening of the inhibitor targeting CPV VP2 protein was verified, and further found that it is through inhibiting the transcription and protein expression level of CPV VP2 gene. Thus, it can prevent CPV virus from invading host cells, and can be used for inhibiting the infection of CPD, and protecting host cells from being damaged by viruses. Therefore, cyproheptadine hydrochloride can be used for treating canine parvovirus disease.
[0012] After the administration of the medicine prepared by the present application with cyproheptadine hydrochloride as the only active ingredient, the temperature, spirit, appetite and fecal state of the puppies with canine parvovirus disease are obviously improved, the cure rate of the medicine of the present application for canine parvovirus disease is 70%, the significant efficiency is 30%, and the total effective rate is 100%; the effect of cyproheptadine hydrochloride of the present application for treating canine parvovirus disease is very outstanding, and the cyproheptadine hydrochloride can be used alone to treat canine parvovirus disease with high efficiency. The cyproheptadine hydrochloride as the only active ingredient has good application prospect in the preparation of the medicine for preventing and / or treating canine parvovirus disease. BRIEF DESCRIPTION OF DRAWINGS
[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.
[0014] Figure 1 TCID after CPV treated by different concentrations of cyproheptadine hydrochloride 50 .
[0015] Figure 2 VP2 mRNA gene transcription level after CPV treated by different concentrations of cyproheptadine hydrochloride.
[0016] Figure 3 Influence on VP2 protein expression after CPV treated by different concentrations of cyproheptadine hydrochloride. DETAILED DESCRIPTION
[0017] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.
[0018] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.
[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.
[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0022] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.
[0023] This invention provides the use of cyproheptadine hydrochloride in the preparation of medicaments for the prevention and / or treatment of canine parvovirus disease.
[0024] In some specific implementations, the canine parvovirus disease refers to canine parvovirus disease caused by canine parvovirus.
[0025] This invention also provides a drug for the prevention and / or treatment of canine parvovirus disease, including cyproheptadine hydrochloride.
[0026] In some specific implementation plans, pharmaceutically acceptable excipients are also included.
[0027] In some specific implementations, the dosage form of the drug is tablets, capsules, granules, emulsions, suspensions, and solutions.
[0028] In some specific implementations, the drug is administered orally.
[0029] Cyproheptadine hydrochloride can inhibit the assembly and release of canine parvovirus and can be used to treat canine parvovirus disease.
[0030] The drug uses cyproheptadine hydrochloride as its active ingredient, with a concentration of 0.1–1000 μM.
[0031] Experimental results show that after applying the drug prepared with cyproheptadine hydrochloride as the sole active ingredient of this invention, the body temperature, mental state, appetite, and fecal condition of puppies suffering from canine parvovirus disease were significantly improved. The drug of this invention has a cure rate of 70%, a significant efficacy rate of 30%, and a total effective rate of 100% for canine parvovirus disease. The cyproheptadine hydrochloride of this invention has a very outstanding effect in treating canine parvovirus disease; it can be used alone to treat canine parvovirus disease with a high efficacy rate. Cyproheptadine hydrochloride, as the sole active ingredient, has good application prospects in the preparation of drugs for the prevention and / or treatment of canine parvovirus disease.
[0032] Cat kidney cells (F81) preserved by the College of Animal Science and Technology of Yangtze University were cultured in DMEM cell culture medium containing 10% fetal bovine serum at 37°C and 5% CO2.
[0033] The CPV virus used in this embodiment of the invention is a canine parvovirus strain from Jingzhou isolated from the intestinal tract of a confirmed deceased dog using F81 cells. This strain was named CPV-JZ2020. The half-cell infection dose (TCID50) of the 5th generation virus on F81 cells was 10. 5.0 / mL. The VP2 gene of 1755bp was amplified by PCR. Compared with the VP2 gene of CPV strain CPV-JZ2020 from domestic and foreign strains, the nucleotide homology was 98.39%-99.83% and the amino acid homology was 97.56%-100.00%. Gene analysis results showed that CPV-JZ2020 strain is CPV-2c type, and the GenBank accession number is MW495851.
[0034] Example 1
[0035] Cyproheptadine hydrochloride against canine parvovirus TCID 50 Impact
[0036] F81 cells (1.5 × 10⁻⁶) were cultured in DMEM cell culture medium containing 10% fetal bovine serum in 12-well plates. 6 Cells were treated with different concentrations of cyproheptadine hydrochloride (0 μM, 10 μM, 100 μM) dissolved in DMSO (<1%) with CPV fluorescently labeled virus (MOI = 0.5). The cell culture plates were then incubated at 37°C and 5% CO2 for 48 h. After three freeze-thaw cycles at -80°C, the cells were used as samples and serially diluted 10-fold with serum-free DMEM cell culture medium to create six dilutions, with each dilution replicated in eight wells. F81 cells were seeded and cultured in the cell culture plates at 37°C and 5% CO2 for 5 days. Fluorescence changes in each well were observed daily, and TCID was calculated. 50 .
[0037] Experimental results are as followsFigure 1 As shown, both 10 μM and 100 μM cyproheptadine hydrochloride significantly reduced the TCID of CPV fluorescently labeled virus. 50 .
[0038] Example 2
[0039] Effects of Cyproheptadine Hydrochloride on Canine Parvovirus VP2 RNA Transcription Levels
[0040] F81 cells (1.5 × 10⁻⁶) were cultured in DMEM cell culture medium containing 10% fetal bovine serum in 12-well plates. 6 Cells were treated with different concentrations of cyproheptadine hydrochloride (0 μM, 10 μM, 100 μM) dissolved in DMSO (<1%) with CPV fluorescently labeled virus (MOI = 0.5). Cell cultures were then incubated at 37°C and 5% CO2 for 48 h, followed by centrifugation and discarding of the supernatant. Total RNA was extracted using the Trizol method, and cDNA was synthesized using the RT Primer Mix kit. RT-qPCR was used to detect differential VP2 gene RNA expression.
[0041] The total volume of the RT-qPCR reaction system was 10 μL, including 0.4 μL of forward and reverse primers, 2 μL of cDNA, 5 μL of SYBR Green (Tiangen), and 2.2 μL of sterile deionized water, bringing the total volume to 10 μL. The reaction conditions were: 95℃ for 2 min; 95℃ for 10 s, 58℃ for 45 s, for 35 cycles.
[0042] The primer sequences for amplifying VP2 are as follows: upstream primer: 5'-CGCGGATCCATGAGTGATGGAGCAGTTC-3'; downstream primer: 5'-CCCAAGCTTTTAATATAATTTTCTAGGTG-3'.
[0043] Experimental results are as follows Figure 2 As shown, cyproheptadine hydrochloride can significantly inhibit the RNA expression level of the CPVVP2 gene.
[0044] Example 3
[0045] Effect of cyproheptadine hydrochloride on VP2 protein levels in CPV
[0046] F81 cells (1.5 × 10⁻⁶) were cultured in DMEM cell culture medium containing 10% fetal bovine serum in 12-well plates. 6Cells were treated with different concentrations of cyproheptadine hydrochloride (0 μM, 10 μM, 100 μM) dissolved in DMSO (<1%) with CPV fluorescently labeled virus (MOI = 0.5). Cell cultures were then incubated at 37°C and 5% CO2 for 48 h, followed by centrifugation and discarding of the supernatant. Total protein was extracted, and differential VP2 protein expression was detected by Western blotting.
[0047] Experimental results are as follows Figure 3 As shown, cyproheptadine hydrochloride can significantly inhibit the expression level of CPVVP2 protein.
[0048] Example 4
[0049] Tablet manufacturing method with cyproheptadine hydrochloride as the main ingredient:
[0050] Add cyproheptadine hydrochloride and starch to a mixer in a weight ratio of 1:2 and mix at 50 rpm for 10 minutes to ensure uniform mixing.
[0051] The mixed powder is added to a granulator for wet granulation. During granulation, the moisture content is controlled between 10% and 15% to ensure the uniformity and flowability of the particles.
[0052] Place the prepared granules into a drying oven, set the temperature to 50℃, and dry for 2 hours until the moisture content of the granules drops below 3%.
[0053] The dried granules are added to a tableting machine, with magnesium stearate added as a lubricant. Tableting is performed at a dosage of 10 mg cyproheptadine hydrochloride per tablet. The pressure is controlled at 10-15 MPa during tableting to ensure tablet hardness and stability.
[0054] Example 5
[0055] Syrup preparation method with cyproheptadine hydrochloride as the main component:
[0056] Weigh 10g of cyproheptadine hydrochloride and add it to 900ml of purified water. Stir until fully dissolved.
[0057] Add 0.15g of sodium saccharin to the solution and stir until well mixed.
[0058] Use a pH adjuster (such as sodium hydroxide or hydrochloric acid) to adjust the pH of the solution to between 6.0 and 6.5.
[0059] Bring the volume to 1L, which is 10mg / ml cyproheptadine hydrochloride syrup.
[0060] The solution is filtered through a filter to remove any insoluble impurities that may be present.
[0061] Experimental Example 1
[0062] 1. Test materials
[0063] 1.1 Drugs
[0064] Cyproheptadine hydrochloride tablets, 10 mg / tablet; Cyproheptadine hydrochloride syrup, 10 mg / ml; starch and sodium saccharin.
[0065] 1.2 Experimental Animals
[0066] Thirty dogs, half male and half female, aged 0.5 to 2 years and weighing 4 to 12 kg, were diagnosed with canine parvovirus disease (CPD) by Changda Pet Hospital in Jingzhou City. All of them showed typical CPD clinical symptoms: fever of 39.9 to 41.3℃, depression, loss of appetite, vomiting, diarrhea, and bloody stools resembling tomato juice.
[0067] 2. Test Methods
[0068] 2.1 Animal grouping
[0069] Thirty confirmed CPD patients were randomly divided into three groups of 10 each: experimental group A, experimental group B, and negative control group.
[0070] 2.2 Animal administration
[0071] Experimental group A was given cyproheptadine hydrochloride tablets orally at a dose of 2 mg / kg twice daily for 7 consecutive days.
[0072] Experimental group B was given cyproheptadine hydrochloride syrup orally at a dose of 2 mg / kg twice daily for 7 consecutive days.
[0073] The control group received oral starch at 4 mg / kg and oral saccharin sodium solution at 0.3 mg / kg.
[0074] 2.3 Clinical symptom observation
[0075] Measure the dog's temperature once in the morning and once in the evening. Observe and record the frequency of vomiting, stool condition, mental state, and food and water intake. Determine and compare the clinical symptoms, disease progression, and treatment effects of sick dogs in each group. Analyze and summarize the results, classify those that have recovered or shown significant improvement as effective cases, and calculate the effectiveness rate.
[0076] 2.4 Efficacy Evaluation Criteria
[0077] (1) Recovery: After taking the medicine, the body temperature is normal, the mental state is good, the appetite is restored, there is no vomiting, the bloody stool stops, and the stool is formed.
[0078] (2) Significant effect: After medication, body temperature, mental state and appetite return to normal, there is no vomiting, bloody stool stops or decreases, and stool is basically formed.
[0079] (3) Ineffective: clinical symptoms do not improve significantly or worsen after medication, or death occurs.
[0080] 3. Test Results
[0081] Table 1. Clinical symptom observation results on day 8 after drug administration.
[0082]
[0083]
[0084] Note: The normal body temperature reference range is 38.5-39.5℃; " / " indicates that the animal is dead.
[0085] Table 2 Treatment effects of each group
[0086]
[0087] Compared with the control group, experimental groups A and B showed significant improvements in body temperature, mental state, thirst, appetite, and stool condition in patients with CPD.
[0088] In summary, this invention provides the use of cyproheptadine hydrochloride as the sole active ingredient in the preparation of drugs for the prevention and / or treatment of canine parvovirus disease. Experimental results show that after administration of the drug prepared with cyproheptadine hydrochloride as the sole active ingredient according to this invention, the body temperature, mental state, appetite, and fecal condition of dogs with parvovirus disease were significantly improved. The drug of this invention has a cure rate of 70% for canine parvovirus disease, a significant efficacy rate of 30%, and a total effective rate of 100%. The cyproheptadine hydrochloride of this invention has a very outstanding effect in treating canine parvovirus disease; it can be used alone to treat canine parvovirus disease with a high efficacy rate. Cyproheptadine hydrochloride as the sole active ingredient has good application prospects in the preparation of drugs for the prevention and / or treatment of canine parvovirus disease.
[0089] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. The use of cyproheptadine hydrochloride in the preparation of drugs for the prevention and / or treatment of canine parvovirus disease.
2. The application according to claim 1, characterized in that, The canine parvovirus disease mentioned refers to canine parvovirus infection caused by canine parvovirus.
3. A drug for the prevention and / or treatment of canine parvovirus disease, characterized in that, Including cyproheptadine hydrochloride.
4. The drug according to claim 3, characterized in that, It also includes pharmaceutically acceptable excipients.
5. The drug according to claim 3, characterized in that, The dosage forms of the drug are tablets, capsules, granules, emulsions, suspensions, and solutions.
6. The drug according to claim 3, characterized in that, The drug is administered orally.